1. Field of the Invention
The present invention relates to anaerobic bacteria belonging to the genus Bifidobacterium useful for gene therapy of solid tumors, a pharmaceutical composition containing the same, a method of delivering a gene and a method of treating solid tumors by use of the same.
2. Description of the Prior Art
Hypoxic regions are characteristic of solid tumors in animal (Int. J. Radiat. Oncol. Biol. Phys., 10: 695-712(1984)) and occur with high frequency in many types of human solid tumors (Fischer-Verlag, stuttgart, 219-232(1994), New York). Tissue oxygen electrode measurements (i.e. a membrane examination device capable of measuring dissolved oxygen) taken in cancer patients have shown a median range of oxygen partial pressure of 10 to 30 mmHg in tumors, with a significant proportion of readings below 2.5 mmHg, whereas those in normal tissues range from 24 to 66 mmHg.
Accordingly, gene therapy in solid tumors that targets gene expression to hypoxic tumor cells is currently being investigated (Nat. Med. 3: 515-520 (1997)). As a result, it is known that certain species of anaerobic bacteria, including the genera Clostridium and Bifidobacterium, can selectively germinate and grow in the hypoxic regions of solid tumors after intravenous (i.v.) injection (Cancer Res. 40: 2061-2068(1980) & 15: 473-478 (1955)).
Further, anaerobic bacteria such as Clostridia or Salmonella have been examined for the availability as gene delivery vectors (Gene Ther. 4: 791-796 (1997) & 3: 173-178 (1996), FEMS Microbiol. Rev. 17:357-364(1995), Cancer Biother Radio. 11: 145-153 (1996), Nat. Biotechnol. 17: 37-41 (1999)).
However, these bacteria have pathogenicity in humans and are thus not always safe gene delivery vectors in gene therapy of solid tumors. Actually, some reports have demonstrated febrile adverse reactions as side effects after injection with Clostridium butyricum spores or oral intake of Salmonella typhi (Eur. J. Cancer. 3: 37-41 (1967), J. Clin. Invest. 90: 412-420 (1992), Infect. Immun. 60: 536-541 (1992)).
The genera Bifidobacterium and Lactobacillus, on the other hand, are Gram-positive and are domestic, nonpathogenic bacteria found in the lower small intestine and large intestine of humans and other animals.
In particular, Bifidobacterium strains have widely used for preparation of fermented dairy products in many Asiatic and Western countries, and it is now generally accepted that these bacteria are nonpathogenic. In addition, it is known that these bacteria are not only nonpathogenic but also have health-promoting properties for their host. Such useful properties include e.g. an increase of the immune response (J. Dairy Sci. 74:1187-1195(1991)), inhibition of carcinogenesis (Cancer Res. 53: 3914-3918 (1993)) and protection of the host against viral infection (Lancet. 344: 1046-1049 (1994)), etc.
Despite the increasing attention to these bacteria in the fields of food science, medicine and industry, they have rarely been used in gene therapy.
To be able to exploit the potential of these bacteria for cancer gene therapy, detailed knowledge is required about such basic biological phenomena as cellular metabolism, gene expression, protein secretion and genetics. But little is known about genetic properties of the genus Bifidobacterium, mainly due to the lack of efficient and reproducible systems for genetic transfer and adequate selectable markers.
In recent years, however, a system for the convenient and reproducible genetic transformation of stains of the genus Bifidobacterium was developed (Microbiology, 142: 109-114 (1996); Biosci. Biotechnol. Biocem. 61: 1211-1212 (1997)).
However, the development of regulatory sequences including a promoter for highly expressing an introduced gene was still not satisfactory.